1. Field of the Invention
The present invention is related to the biotechnology field, in particular with new recombinant antibodies obtained by genetic engineering, specifically with chimeric and humanized antibodies obtained from the murine monoclonal antibody P3 (MAb P3) and its anti-idiotype murine monoclonal antibody 1E10 (MAbai 1E10).
More specifically, the invention is related with antibodies that bind to gangliosides containing N-glycolylated sialic acid, but not with the acetylated forms of the gangliosides or with neuter glycolipids. Gangliosides containing N-glycolylated sialic acid are antigens widely expressed in breast cancer and melanomas. On the other hand, the anti-tumor effect of the MAbai 1E10 has also been demonstrated in experimental models.
The present invention is also related with the pharmaceutical compositions that contain the previously described recombinant antibodies useful in the diagnosis and therapy of cancer, particularly breast cancer and melanomas.
2. Discussion of the Prior Art
Gangliosides are glycosphingolipids that contain sialic acid and they are present in the plasmatic membrane of cells in vertebrates (StuIts et al. (1989):
Glycosphingolipids: structure, biological source and properties, Methods Enzymology, 179:167-214). Some of these molecules have been reported in the literature as tumor-associated antigens or tumor markers (Hakomori et al. (1991): Possible functions of tumor associated carbohydrate antigens, Curr. Opin. Immunol., 3: 646-653). For that reason the use of anti-ganglioside antibodies has been described as useful in the diagnosis and therapy of cancer (Hougton et al. (1985): Mouse monoclonal antibody IgG3 antibody detecting GD3 ganglioside: to phase I trial in patients with malignant melanoma, PNAS USA, 82:1242-1246; Zhang et al. (1997): Selection of carbohydrate tumor antigens as targets for immune attack using immunohistochemistry. I. Focus on gangliosides, Int. J. Cancer, 73: 42-49).
The sialic acids more frequently expressed in animals are N-acetyl (NeuAc) and N-glycolyl (NeuGc) (Corfield et al. (1982): Occurrence of sialic acids, Cell. Biol. Monogr., 10: 5-50). Generally, NeuGc is not expressed in normal human and chickens tissues, but it is broadly distributed in other vertebrates (Leeden and Yu, (1976): Chemistry and analysis of sialic acid. In: Biological Role of Sialic Acid. Rosemberg A and Shengtrund C L (Eds). Plenum Press, New York, 1-48; Kawai et al. (1991): Quantitative determination of N-glycolylneuraminic acid expression in human cancerous tissues and avian lymphoma cell lines as a tumor associated sialic acid by gas chromatography-mass spectrometry, Cancer Research, 51: 1242-1246). However, there are reports that show that anti-NeuGc antibodies recognize some human tumors and tumor cell lines (Higashi et al. (1988): Detection of gangliosides as N-glycolylneuraminic acid specific tumor-associated Hanganutziu-Deicher antigen in human retinoblastoma cells, Jpn. J. Cancer Res., 79: 952-956; Fukui et al. (1989): Detection of glycoproteins as tumor associated Hanganutziu-Deicher antigen in human gastric cancer cell line, NUGC4, Biochem. Biophys. Res. Commun., 160: 1149-1154). Increased levels of GM3 (NeuGc) gangliosides have been found in human breast cancer (Marquina et al. (1996): Gangliosides expressed in human breast cancer, Cancer Research, 1996; 56: 5165-5171), and this result makes attractive the use of this molecule as a target for cancer therapy.
The monoclonal antibody (Mab) P3, produced by the cell line deposited with accession number ECACC 94113026 (European Patent EP 0 657 471 B1), is a murine monoclonal antibody with IgM isotype. Mab P3 was obtained when fusing murine splenocytes from a BALB/c mouse immunized with liposomes containing GM3(NeuGc) and tetanic toxoid with the cell line P3-X63-Ag8.653, which is a murine myeloma. This Mab P3 reacts strongly with N-glycolylated sialic acid-containing gangliosides but not with the acetylated forms of the gangliosides, nor with the neuter glycolipids. It was demonstrated by immunocytochemical and immunohistochemical studies carried out with cell lines and tissues from benign and neoplasic tumors that the Mab P3 recognizes breast cancer (Vzquez et al. (1995): Generation of a murine monoclonal antibody specific for N-glycolylneuraminic acid-containing gangliosides that also recognizes sulfated glycolipids, Hybridoma, 14: 551-556) and melanoma.
The Mab P3 induced an anti-idiotypic immune response (Ab2) in BALB/c mice (syngeneic model), even without adjuvant and carrier protein (Vazquez et al. (1998): Syngeneic anti-idiotypic monoclonal antibodies to an anti-NeuGc-containing ganglioside monoclonal antibody, Hybridoma, 17: 527-534). A role for the electronegative groups, sialic acid (for gangliosides) or SO3— (for sulfatides), in the recognition properties of this antibody was suggested by immunochemical analysis (Moreno et al. (1998): Delineation of epitope recognized by an antibody specific for N-glycolylneuraminic acid-containing gangliosides, Glycobiology, 8: 695-705).
The anti-idiotypic Mab 1E10 (Mabai 1E10) of IgG1 subtype was obtained from a BALB/c mouse immunized with the Mab P3 coupled to KLH (U.S. Pat. No. 6,063,379, cell line deposited under accession number ECACC 97112901). Mabai 1E10 specifically recognized MAb P3 and did not bind other IgM anti-ganglioside antibodies. Moreover, Mabai 1E10 inhibited the specific binding of Mab P3 to GM3(NeuGc) and to the ductal breast carcinoma-derived cell line MDA-MB-435, which is positive for Mab P3 binding. The MAbai 1E10 induced a strong immune response of Ab3 antibodies when mice from syngeneic or alogenic models were immunized. These Ab3 antibodies did not exhibit the same specificity as the Mab P3 even though they carry idiotopes similar to those carried by the Ab1 antibody (Vazquez et al. (1998): Syngeneic anti-idiotypic monoclonal antibodies to an anti-NeuGc-containing ganglioside monoclonal antibody, Hybridoma, 17: 527-534). MAbai 1E10 induced a strong antitumor effect in syngeneic as well as alogenic mice. The growth of the mammary carcinoma cell line F311 was significantly reduced by repeated doses of KLH-coupled MAbai 1E10 in Freund's adjuvant when BALB/c mice were vaccinated. Also the number of spontaneous lung metastasis was reduced after the vaccination. Intravenous administration of the MAbai 1E10 to C57BLU6 mice, 10 to 14 days after the intravenous inoculation of B16 melanoma cells, caused a dramatic reduction of the number of lung metastases when compared with mice treated with an irrelevant IgG. These results suggest that more than one anti tumor effect mechanism is triggered (Vazquez et al. (2000): Anti tumor properties of an anti-idiotypic monoclonal antibody in relation to N-glycolyl-containing gangliosides, Oncol. Rep., 7: 751-756, 2000).
Even though hybridoma technology has been developed for 15 years (Koehler y Milstein (1975): Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 256: 495497) and monoclonal antibodies are still very useful in diagnosis as well as research, they have not demonstrated their therapeutic effectiveness in humans. This has been mainly due to their short half-life in blood, to the human anti-mouse antibody immune response (HAMA response), and also because murine effector functions fail for the human immune system.
Genetic engineering technology has revolutionized MAb potential, since by manipulating immunoglobulin genes it is possible to obtain modified antibodies with reduced antigenicity, as well as to improve their effector functions when used in the treatment or diagnosis of certain pathologies. Methods for reducing immunoglobulin immunogenicity have as essential object to diminish the differences between the murine antibody and a human immunoglobulin, without altering the antigen recognition specificity (Morrison y Oi (1989): Genetically engineered antibody molecules, Adv Immunol., 44: 65-92).
Recently, several methods have been developed to humanize murine or rat antibodies, thus reducing the xenogenic immune response against foreign proteins when they are injected into humans. One of the first approaches to reduce the antigenicity were chimeric antibodies, in which the variable domains of the murine protein are inserted in constant domains of human molecules that exhibit the same specificity but reduced immunogenicity compared to their murine counterparts. Additionally, human effector functions are preserved by chimeric antibodies (Morrison et al. (1984): Chimeric human antibody molecules: Mouse antigen-binding domains with human constant region domains, PNAS USA, 81: 6851-6855). Even when chimeric antibodies have the same specificity as their murine counterpart, an immune response to the rodent variable regions is frequently observed.
In an attempt to further reduce the immunogenicity of chimeric antibodies, only the CDRs from the rodent monoclonal antibody have been grafted onto human framework regions and this hybrid variable region has been expressed with human constant regions (Jones et al. (1986): Replacing the complementary-determining regions in a human antibody with those from a mouse, Nature 321: 522-524; Verhoeyen et al. (1988): Reshaping human antibodies: grafting an antilysozyme activity, Science 239, 1534-1536). However, this approach has several shortcomings: frequently the resulting antibody has decreased affinity and a number of framework residues must be mutated back to the corresponding murine ones to restore binding (Rietchmann et al. (1988): Reshaping human antibodies for therapy, Nature, 332: 323-327; Queen et al. (1989): A humanized antibody that binds to the interleukin 2 receptor, PNAS USA, 86: 10029-10033; Tempest et al. (1991): Reshaping a human monoclonal antibody to inhibit human respiratory syncytial virus infection in vivo, Biotechnology, 9: 266-272). In addition, persisting immunogenicity is frequently observed in the CDR-grafted antibodies.
Mateo and collaborators (U.S. Pat. No. 5,712,120) have described a procedure for reducing immunogenicity of murine antibodies. According to the method, the modifications are restricted to the variable domains and specifically to the murine FRs of chimeric antibodies. Moreover, the replacements are only carried out in those regions of the FRs that have amphipatic sequences and therefore they are potential epitopes recognized by T cells.
The method comprises judicious replacement of a few amino acid residues, located in the potential immunogenic epitopes by the corresponding residues from the most homologous human sequence. Those amino acids that are mainly responsible for canonical structures, as well as the residues in the immediate neighborhood of the CDRs or in the Vernier zone must be retained.
The resulting antibody retains its antigen binding specificity and is less immunogenic than either its murine or chimeric predecessor (Mateo et al. (2000): Removal of T cell epitopes from genetically engineered antibodies: Production of modified immunoglobulins with reduced immunogenicity, Hybridoma 19: 463-71). These properties increase its therapeutic usefulness. Using this new procedure, only few mutations, and of course less genetic manipulations, have to be done.